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Elod Kortvely, Sascha Dammeier, Michelle Dierstein, Franziska Klose, Katja Schenke-Layland, Marius Ueffing; Use of laser capture microdissection coupled mass spectrometry to analyze the choroidal proteome relevant to AMD. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5807.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is a sight-threatening disorder of the central retina. Emerging evidence suggests that the loss of choroidal vasculature may be the initial insult leading to the dysfunction of photoreceptors, but the underlying molecular mechanisms are still to be elucidated. We performed a systematic analysis of the human choroidal proteome to gain insights into underlying disease processes.
Human post-mortem eyes from six donors were removed and fixed with cross-linking (10% buffered formalin) fixative. Laser capture microdissection was used to isolate minute amounts of the regions of interest from cresyl violet stained sections for subsequent proteomics analysis. Catapulted tissue patches were directly collected in CapSure® caps and subjected to alternating cycles of ambient to high pressure (up to 35,000 psi), in order to facilitate enzymatic digestions of samples. Mass spectrometry analysis was conducted after trypsinization of the samples. Photoreceptor outer segments were also isolated along with choroidal samples serving as controls to detect abundant proteins also present outside the choroid. LC-MS/MS analysis was performed an Orbitrap Fusion mass spectrometer. All acquired spectra were analyzed using the MaxQuant software.
Formalin fixation gave satisfactory results in preserving tissue morphology and protein identification. Hundred eighty-four proteins were identified both in choroidal and in retinal samples, while thirty proteins were specific to choroid. As expected, most of the choroid-specific proteins are constituents of the extracellular matrix (e.g. Heparan Sulfate Proteoglycan 2, lamininins) and also collagens with known AMD risk variants in the corresponding genes (COL4A3, COL8A1). Besides, melanocyte-specific proteins (e.g. Transmembrane Glycoprotein NMB) were also characteristic for the choroid.
The combination of LCM and mass spectrometric technologies may be effective in detecting protein profiles within the choroid specific to age-related changes and to certain pathologies. Using high-pressure assisted sample preparation not only abridged processing, but vastly improved protein identification offering great analytical depth. This strategy can contribute to a better understanding of the role of choroid in the pathomechanism of AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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